Methods and compositions for treating and preventing distal bowel lesions

ABSTRACT

This invention features compositions and methods for treating and preventing distal bowel disease by rectal administration of intestinal trefoil factors. The intestinal trefoil factor can be administered either alone or in combination with one or more antimicrobial agents, anti-inflammatory agents or analgesics.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of the filing date of U.S.Provisional Application No. 60/309,238 (filed Jul. 31, 2001), herebyincorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The invention features methods for treating lesions of the distalbowel using intestinal trefoil peptides.

[0003] Proctitis and enteritis involve the destruction of the smalland/or large bowel epithelium, resulting in erythema, ulcerations,diarrhea, bleeding from the bowel, malabsorption of nutrients and,frequently severe abdominal pain. Enteritis and proctitis often arise asa complication of antineoplastic therapy, such as cancer chemotherapy orradiotherapy directed at the abdomen or pelvic area. This syndrome mayalso result from natural diseases like Crohn's disease, ulcerativecolitis, vascular insufficiency, infection, allergic conditions, or fromother causes that are less well understood. The painful ulcerativelesions of enteritis or proctitis can cause patients to restrict theirfood and liquid intake, resulting in weight loss and dehydration. Severeenteritis can necessitate the de-escalation or the complete interruptionof a planned chemo/radio-therapeutic dosing.

[0004] Secondary infections by pathogenic microorganisms are a seriousconsequence of the mucosal damage caused by enteritis and proctitis.These conditions, when severe, are risk factors for chronic debilitatinglocal infections (e.g., yeast (Candida spp.) infections) andsepticemias. The microorganisms use the compromised bowel epithelium asa portal of entry into the body. The problem of secondary infection ismade more serious by the immunocompromised status of patients undergoingcancer treatment (chemotherapy or radiotherapy).

[0005] The overall frequency of enteritis and proctitis varies with thepatient's diagnosis, age, and general level of health, as well as thetype, dose, and frequency of cancer therapy. Approximately 40% of allpatients who receive chemotherapy suffer some degree of mucositis of thegastrointestinal tract. Enteritis or proctitis occur in the majority ofpatients undergoing either total abdominal radiation or high doseradiotherapy to the perineal area.

[0006] Vascular, particularly arterial, insufficiency of the bowel is arelatively common and serious problem in patients suffering fromatherosclerotic disease, but may also occur in disease-free patients.Reduced blood flow to the gastrointestinal tract can result inepithelial cell loss, making the patient susceptible to malabsorptiveconditions and secondary microbial infections.

SUMMARY OF THE INVENTION

[0007] This invention features methods and compositions for treating orpreventing a lesion of the distal bowel in a mammal by administering, tothe rectum of the mammal, a therapeutically effect amount of anintestinal trefoil peptide. Treatment or prevention of lesions accordingto the invention can speed healing, reduce pain, delay or preventoccurrence of the lesion, and inhibit expansion, secondary infection, orother complications of the lesion. Preferably, the mammal is a human. Inparticularly useful embodiments, the intestinal trefoil peptide is SP,pS2, ITF, ITF₁₅₋₇₃, ITF₂₁₋₇₃, ITF₁₋₇₂, ITF₁₅₋₇₂, or ITF₂₁₋₇₂, and ispresent in a pharmaceutical composition containing a pharmaceuticallyacceptable carrier. Other useful intestinal trefoil peptides includepolypeptides that are substantially identical to SP, pS2, ITF, ITF₁₅₋₇₃,ITF₂₁₋₇₃, ITF₁₅₋₇₂, or ITF₂₁₋₇₂. Preferably, the intestinal trefoilpeptide is ITF, which may be administered as a monomer, a dimer, oranother multimeric form.

[0008] The methods and compositions of this invention are particularlyuseful for treating lesions of the distal bowel such as proctitis,enteritis, Crohn's Disease, ulcerative colitis, lesions caused byphysical trauma or surgical intervention (e.g., biopsy, resection, orhemorrhoidectomy), or lesions caused by antineoplastic therapy (e.g.,chemotherapy or radiation therapy). Additionally, lesions of the distalbowel that result from microbial (e.g., bacterial, viral, or fungal)infection are also amenable to treatment.

[0009] In preferred embodiments of the methods and compositions, asecond therapeutic agent is included. Desirable second therapeuticagents include anti-inflammatory agents, antibacterial agents (e.g.,penicillins, cephalosporins, tetracyclines, or aminoglycosides),antifungal agents (e.g., nystatin or amphotericin B), antiviral agents(e.g., acyclovir), analgesics (e.g., lidocaine or benzocaine), orsteroids (e.g., triamcinolone, budesonide, or hydrocortisone). Otherparticularly useful second therapeutic agents include 5-aminosalicylicacid derivatives such as sulfasalazine, mesalamine, olsalazine, andbalsalazide, anti-TNF-α monoclonal antibodies such as infliximab(Remicade®), and other drugs useful for treating lesions of the distalbowel such as metronidazole. The second therapeutic agent may beadministered within (either before or after administration of theintestinal trefoil peptide) 14 days, 7 days, 1 day, 12 hours, 1 hour, orsimultaneously with the intestinal trefoil peptide.

[0010] The second therapeutic agent can be present in the same ordifferent pharmaceutical compositions as the intestinal trefoil peptide.When the second therapeutic agent is present in a differentpharmaceutical composition, different routes of administration may beused. For example, the second therapeutic may be administered orally, orby intravenous, intramuscular, or subcutaneous injection. The secondtherapeutic need not be administered rectally.

[0011] Suitable pharmaceutical compositions include at least anintestinal trefoil peptide and a pharmaceutically acceptable carrier.Particularly useful pharmaceutical compositions contain bioerodablemicrospheres that encapsulate one or more of the therapeutic agents. Inother useful embodiments, a mucoadhesive or viscosity-enhancing agent ispresent. Alternatively, the trefoil peptide can be formulated fortopical application as a concentrated paste to be applied directly tothe lesion via a pledget and stick applicator. Rectal administration ofthe trefoil peptide may be supplemented by oral administration the sameor a different trefoil peptide.

[0012] Mammalian intestinal trefoil peptides were discovered in 1982.One of the mammalian intestinal trefoil peptides, human intestinaltrefoil factor (ITF; TFF3), has been characterized extensively, and isdescribed in U.S. Pat. Nos. 6,063,755, and 6,221,840, herebyincorporated by reference. The other two known human intestinal trefoilpeptides are spasmolytic polypeptide (SP; TFF2) and pS2 (TFF1).Intestinal trefoil peptides, described extensively in the literature(e.g., Sands et al., Annu. Rev. Physiol. 58: 253-273, 1996), areexpressed in the gastrointestinal tract and have a three-loop structureformed by intrachain disulfide bonds between conserved cysteineresidues. These peptides protect the intestinal tract from injury andcan be used to treat intestinal tract disorders such as peptic ulcersand inflammatory bowel disease. Homologs of these human peptides havebeen found in a number of non-human animal species. All members of thisprotein family, both human and non-human, are referred to herein asintestinal trefoil peptides. Human ITF will be referred to mostextensively in this application; however, the activity of human ITF iscommon to each of the mammalian intestinal trefoil peptides.

[0013] “Intestinal trefoil peptide,” as used herein, includes allmammalian homologs of human spasmolytic polypeptide (SP; also known asTFF2), human pS2 (also known as TFF1) and human intestinal trefoilfactor (ITF; also known as TFF3), and biologically active fragmentsthereof. Homologs of the trefoil peptides have, preferably, 70% aminoacid identity to the human sequence, more preferably 85% identity, mostpreferably 95%, or even 99% sequence identity. The length of comparisonsequences will generally be at least about 10 amino acid residues,usually at least 20 amino acid residues, more usually at least 30 aminoacid residues, typically at least 45 amino acid residues, and preferablymore than 60 amino acid residues. Alternatively, intestinal trefoilpeptides are polypeptides encoded by a polynucleotide that hybridizeswith high stringency to the human ITF, pS2, or SP cDNAs provided in SEQID NOs: 4, 5, and 6, respectively, or the human ITF, pS2, or SP genesprovided in SEQ ID NOs: 7, 8, and 9, respectively.

[0014] The term “fragment” is meant to include polypeptides that aretruncations or deletions of SP, pS2 and ITF. Preferably, the fragmentshave 70% amino acid identity to the corresponding regions of the humanpolypeptide sequence. More preferably, the fragments are 85% identical,most preferably 95%, or even 99% identical to the human polypeptidesequence to which they correspond. The length of comparison sequenceswill generally be at least about 10 amino acid residues, usually atleast 20 amino acid residues, more usually at least 30 amino acidresidues, typically at least 45 amino acid residues, and preferably morethan 60 amino acid residues.

[0015] Preferable fragments contain four cysteine residues in anypositions which correspond to the cysteines at positions 25, 35, 45, 50,51, 62, or 71, of human ITF (FIG. 1), or positions 31, 41, 51, 56, 57,68, and 82 of human pS2 (FIG. 2). More preferably, fragments containfive cysteine residues at these positions. Most preferably, six, or evenall seven cysteines are present.

[0016] Fragments of SP are meant to include truncations or deletions andpreferably have 70% sequence identity to the corresponding human SPpolypeptide sequence (FIG. 3). More preferably, the fragments are 85%identical, most preferably 95%, or even 99% identical to the humanpolypeptide sequence. Preferably, active fragments contain at least fourcysteine residues which correspond to positions 6, 8, 19, 29, 34, 35,46, 58, 68, 78, 83, 84, 95, and 104 in the human SP polypeptide. Morepreferably, fragments contain six cysteines which correspond to thesepositions. Even more preferable are fragments that contain eightcysteines. Most preferable are fragments that contain cysteines at ten,twelve, or even, all fourteen positions.

[0017] It is recognized in the art that one function of the identifiedcysteine residues is to impart the characteristic three-loop (trefoil)structure to the protein. Accordingly, preferred fragments of ITF andpS2 have a least one loop structure, more preferably, the fragments havetwo loop structures, and most preferably, they have three loopstructures. It is equally well recognized that the native SP polypeptidehas a six loop confirmation. Preferable fragments contain at least twoof these loop structures, more preferably, four loop structures areconserved, and most preferably, five, or even all six loop structuresare present.

[0018] By “co-formulated” is meant any single pharmaceutical compositionwhich contains two or more therapeutic or biologically active agents.

[0019] By “pharmaceutical preparation” or “pharmaceutical composition”is meant any composition which contains at least one therapeutically orbiologically active agent and is suitable for administration to apatient. For the purposes of this invention, pharmaceutical compositionssuitable for delivering a therapeutic to the distal bowel include, butare not limited to suppositories, enemas, and pastes. Any of theseformulations can be prepared by well known and accepted methods of art.See, for example, Remingtion: The Science and Practice of Pharmacy,19^(th) edition, (ed. AR Gennaro), Mack Publishing Co., Easton, Pa.,1995.

[0020] By “therapeutically effective amount” is meant an amountsufficient to provide medical benefit. When administering trefoilpeptides to a human patient according to the methods described herein, atherapeutically effective amount is usually about 1-2500 mg ofintestinal trefoil peptide per dose. Preferably, the patient receives,10 mg, 100 mg, 500 mg, 750 mg, 1000 mg, 1500 mg, or 2000 mg ofintestinal trefoil peptide in each dose. Dosing is typically performed1-5 times each day.

[0021] By “distal bowel” is meant the portion of the digestive systemthat includes the ascending, transverse, descending, and sigmoid colon,rectum, and anal sphincter.

[0022] By “biologically active,” when referring to an intestinal trefoilpeptide, fragment, or homolog is meant any polypeptide that exhibits anactivity common to its related, naturally occurring family member, andthat the activity is common to the family of naturally occurringintestinal trefoil peptides. An example of a biological activity commonto the family of trefoil peptides is the ability to altergastrointestinal motility in a mammal.

[0023] By “isolated DNA” is meant DNA that is free of the genes which,in the naturally-occurring genome of the organism from which the givenDNA is derived, flank the DNA. Thus, the term “isolated DNA”encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA.

[0024] By “treating” is meant administering a pharmaceutical compositionfor prophylactic and/or therapeutic purposes. The active ingredients ofthe pharmaceutical composition can treat the primary indication (e.g.,epithelial lesion) or secondary symptoms (e.g., concomitant infection,pain, or inflammation).

[0025] By “analgesic” is meant an agent which relieves pain by elevatingthe pain threshold without significantly disturbing the consciousness ofthe patient.

[0026] By “antimicrobial agent” is meant any compound that alters thegrowth of bacteria or fungi cells, or viruses whereby growth isprevented, stabilized, or inhibited, or wherein the microbes are killed.In other words, the antimicrobial agents can be microbiocidal ormicrobiostatic.

[0027] By “antineoplastic therapy” is meant any treatment regimen usedto treat cancer. Typical antineoplastic therapies include chemotherapyand radiation therapy.

[0028] By “substantially identical” is meant a polypeptide or nucleicacid exhibiting at least 75%, but preferably 85%, more preferably 90%,most preferably 95%, or 99% identity to a reference amino acid ornucleic acid sequence. For polypeptides, the length of comparisonsequences will generally be at least 20 amino acids, preferably at least30 amino acids, more preferably at least 40 amino acids, and mostpreferably 50 amino acids. For nucleic acids, the length of comparisonsequences will generally be at least 60 nucleotides, preferably at least90 nucleotides, and more preferably at least 120 nucleotides.

[0029] By “high stringency conditions” is meant any set of conditionsthat are characterized by high temperature and low ionic strength andallow hybridization comparable with those resulting from the use of aDNA probe of at least 40 nucleotides in length, in a buffer containing0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65 C, or a buffer containing 48% formamide, 4.8×SSC, 0.2M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10% dextran sulfate, and 0.1%SDS, at a temperature of 42° C. Other conditions for high stringencyhybridization, such as for PCR, Northern, Southern, or in situhybridization, DNA sequencing, etc., are well-known by those skilled inthe art of molecular biology. See, e.g., F. Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998,hereby incorporated by reference. Other features and advantages of theinvention will be apparent from the following detailed description, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is an amino acid sequence of a human intestinal trefoilfactor (ITF; Accession No. BAA95531; SEQ ID NO: 1).

[0031]FIG. 2 is an amino acid sequence of a human pS2 protein (AccessionNo. NP_(—)003216; SEQ ID NO:2).

[0032]FIG. 3 is an amino acid sequence of human spasmolytic polypeptide(SP; Accession No. 1909187A; SEQ ID NO:3).

[0033]FIG. 4 is a cDNA sequence encoding a human intestinal trefoilfactor (SEQ ID NO:4).

[0034]FIG. 5 is a cDNA sequence encoding a human pS2 protein (SEQ IDNO:5).

[0035]FIG. 6 is a cDNA sequence encoding a human spasmolytic polypeptide(SEQ ID NO:6).

[0036]FIG. 7 is the nucleotide sequence of a gene encoding humanintestinal trefoil factor (locus 10280533:52117-55412; SEQ ID NO:7).

[0037]FIG. 8 is the nucleotide sequence of a gene encoding human pS2protein (locus 10280533:16511-21132; SEQ ID NO:8).

[0038]FIG. 9 is the nucleotide sequence of a gene encoding humanspasmolytic polypeptide (locus 10280533:957-5208; SEQ ID NO:9).

DETAILED DESCRIPTION

[0039] The invention provides methods and compositions useful for thetreatment of a wide range of lesions of the distal bowel. Lesionsamenable to treatment using the methods and compositions of thisinvention include epithelial lesions of the anal sphincters, rectum, andcolon, particularly the sigmoid colon and the descending colon. Theselesions are treated by local application of intestinal trefoil peptideseither alone or in combination with a second therapeutic agent and maybe administered by any delivery device that is useful for deliveringtherapeutics to the distal bowel.

[0040] Pharmaceutical Preparations

[0041] Enemas

[0042] The enemas used to deliver the intestinal trefoil peptides ofthis invention are retention enemas, not evacuation enemas. Enemas, whenadministered in volumes of less than about 50 ml, deliver therapeuticsto the rectum and sigmoid colon. However, enema volumes of about 150-250ml can be used to deliver therapy to the descending, transverse and, insome cases, the ascending colon.

[0043] ITF-Containing Enema for Treatment of Ulcerative ColitisIntestinal trefoil factor 750 mg Sulfasalazine  3 grams Distilled water250 ml

[0044] ITF-Containing Bulk Enema Suspension Suitable for RefrigerationIntestinal trefoil factor 5 g/L 5-aminosalicylic acid 42 g/L NaH₂PO₄ 0.4g/L Na₂HPO₄ 4.48 g/L NaCl 9 g/L Sodium ascorbate 0.5 g/L Tragacanth 4g/L Methylparaben 2 g/L Propylparaben 0.5 g/L Propylene glycol 25 ml/LDistilled water

[0045] Suppositories

[0046] Suppositories are solid dosage forms for insertion into therectum for delivering medication to the rectum and sigmoid colon.Typically, after insertion, the suppository softens, melts, disperses,or dissolves in the lumenal fluid. Rectal suppositories for adults areusually about 2-5 grams each and tapered on both ends. Infantsuppositories are usually about half the size of the adult formulations.

[0047] Either a fatty or a water soluble/water miscible suppository basecan be used in the compositions of this invention. Suitable fatty basesinclude, for example, cocoa butter, theobroma oil, vegetable oilsmodified by esterification, hydrogenation, glycerinated gelatin and highmolecular weight polyethylene glycols. Sustained release and/orprolonged contact of the therapeutics can be achieved by properselection of a fatty suppository base material. Cocoa butter, forexample, melts quickly at body temperature but is immiscible with bodyfluids, resulting in a prolonged but low level delivery of fat-solubletherapeutics to the affected sites. Alternatively, water soluble orwater miscible bases (e.g., polyethylene glycols and glycol-surfactantmixtures) typically dissolve or disperse quickly, resulting in a rapiddelivery of the therapeutic to the affected sites. An exemplarysuppository formulation is provided below.

[0048] ITF-Containing Suppository Tablet Intestinal trefoil factor 300mg Polyethylene glycol 1000  96% Polyethylene glycol 4000  4%

[0049] This formulation has a low-melting point and may requirerefrigeration to maintain in a solid state. Because the intestinaltrefoil peptides are proteinaceous, refrigeration may be desirable. Thelow melting point of the formulation results in rapid suppositorymelting following insertion, resulting in greater patient comfort. Ifrefrigeration is not possible, or if heat molding techniques are used,the amount of polyethylene glycol 4000 may be increased to achieve asufficiently heat stable formulation.

[0050] In an alternative formulation, the intestinal trefoil peptidesand/or other therapeutics can be encapsulated in bioerodablemicrospheres rather than being dissolved in the aqueous phase of theformulation. A wide variety of microencapsulation drug delivery systemshave been developed and many share similar polymeric compositions asused for bioerodable films. Polymers commonly used in the formation ofmicrospheres include, for example, methylacrylate polymers,poly-ε-caprolactone, poly(ε-caprolactone-Co-DL-lactic acid),poly(DL-lactic acid), poly(DL-lactic acid-Co-glycolic acid) andpoly(ε-caprolactone-Co-glycolic acid) (see, for example, Pitt et al., J.Pharm. Sci., 68:1534, 1979).

[0051] Microspheres can be made by procedures well known in the artincluding spray drying, coacervation, and emulsification (see forexample Davis et al. Microsphere and Drug Therapy, Elsevier, 1984;Benoit et al. Biodegradable Microspheres: Advances in ProductionTechnologies, Chapter 3, Ed. Benita, S, Dekker, New York, 1996;Microencapsulation and Related Drug Processes, Ed. Deasy, Dekker, 1984,New York; U.S. Pat. No. 6,365,187). Preferably, the microspheres arebioadhesive or are prepared in formulations containing a bioadhesiveexcipient.

[0052] Other technical features of the intestinal trefoilpeptide-containing solutions are easily modified to suit the specificpharmaceutical formulation and the clinical indication being treated.For example, the pH and osmolarity of the formulation may be adjusted toconfer trefoil peptide stability, while minimizing gastrointestinalirritancy and sensitivity.

[0053] Ointments, Pastes, and Gels

[0054] Lesions of the epithelium of the anal sphincter and thesurrounding skin, such as those resulting from trauma or hemorrhoids,are amenable to trefoil peptide therapy delivered as an ointment, paste,or gel. The viscous nature of these types of preparations allows fordirect application into the wound site. Optionally, the wound site canbe covered with a dressing to retain the trefoil peptide-containingcomposition, protect the lesion and/or absorb exudate. As discussedfurther below, these preparations are particularly useful to restoreepithelial integrity following traumatic surgical procedures (e.g.,hemorrhoidectomy). Such viscous formulations may also have a localbarrier effect thereby reducing irritation and pain.

[0055] Mucoadhesives

[0056] A mucoadhesive excipient can be added to any of the previouslydescribed pharmaceutical compositions. The mucoadhesive formulationscoat the lesioned area, resulting in retention of the intestinal trefoilpeptide at the lesion site, providing protection, inhibiting irritation,and accelerating healing of inflamed or damaged tissue. Mucoadhesiveformulations suitable for use in these pharmaceutical preparations arewell known in the art (e.g., U.S. Pat. No. 5,458,879). Particularlyuseful mucoadhesives are hydrogels composed of about 0.05-20% of awater-soluble polymer such as, for example, poly(ethylene oxide),poly(ethylene glycol), poly(vinyl alcohol), poly(vinyl pyrrolidine),poly(acrylic acid), poly(hydroxy ethyl methacrylate), hydroxyethyl ethylcellulose, hydroxy ethyl cellulose, chitosan, and mixtures thereof.These polymeric formulations can also contain a dispersant such assodium carboxymethyl cellulose (0.5-5.0%).

[0057] Other preferred mucoadhesive excipients for liquid compositionsare ones that allow the composition to be administered as a flowableliquid but will cause the composition to gel in the distal bowel,thereby providing a bioadhesive effect which acts to hold thetherapeutic agents at the lesion site for an extended period of time.The anionic polysaccharides pectin and gellan are examples of materialswhich when formulated into a suitable composition will gel in the distalbowel, owing to the presence of cations in the mucosal fluids. Theliquid compositions containing pectin or gellan will typically consistof 0.01-20% w/v of the pectin or gellan in water or an aqueous buffersystem.

[0058] Other useful compositions which promote mucoadhesion andprolonged therapeutic retention in the distal bowel are colloidaldispersions containing 2-50% colloidal particles such as silica ortitanium dioxide. Such formulations form as a flowable liquid with lowviscosity suitable as an enema; however, the particles interact withglycoprotein, especially mucin, transforming the liquid into a viscousgel, providing effective mucoadhesion (e.g., U.S. Pat. Nos. 5,993,846and 6,319,513).

[0059] Therapeutics Agents

[0060] Trefoil Peptides

[0061] The therapeutic intestinal trefoil peptide(s) are typicallymammalian intestinal trefoil peptides. Preferably, human intestinaltrefoil peptides are used; however, trefoil peptides from other speciesincluding rat, mouse, and non-human primate, may be used. Typically, theintestinal trefoil peptide is intestinal trefoil factor (ITF); however,spasmolytic polypeptide (SP), or pS2 are also useful.

[0062] The intestinal trefoil peptides are administered at 1-5000 mg perdose, preferably 5-2500 mg per dose, or more preferably 10-1500 mg perdose, depending on the nature and condition of the lesion being treated,the anticipated frequency and duration of therapy, and the type ofpharmaceutical composition used to deliver the trefoil peptide. Theintestinal trefoil peptides are typically administered 1-5 times perday.

[0063] Therapeutic Fragments of Intestinal Trefoil Factor (ITF)

[0064] We have also discovered that particular ITF fragments retainbiological activity and may be substituted in any method or compositionin which ITF is used. Methods and compositions containing ITF, in whichthese ITF fragments may be substituted, are described, for example, inU.S. Pat. Nos. 6,063,755 and 6,221,840, and U.S. patent application Ser.No. 10/131,363, filed Apr. 24, 2002, No. 60/317,657, filed Sep. 6, 2001,No. 60/327,673, filed Oct. 5, 2002, No. 60/333,836, filed Nov. 28, 2001,and No. 60/367,574, filed Mar. 26, 2002 (hereby incorporated byreference).

[0065] Particularly useful ITF fragments that retain biological activityinclude the polypeptide corresponding to amino acid residues 15-73 ofSEQ ID NO: 1 (ITF₁₅₋₇₃) and amino acid residues 21-73 of SEQ ID NO: 1(ITF₂₁₋₇₃). Other useful ITF fragments are formed following cleavage ofthe C-terminal phenylalanine residue (i.e., ITF₁₋₇₂, ITF₁₅₋₇₂, andITF₂₁₋₇₂).

[0066] The biologically active ITF fragments of this invention can beproduced using any appropriate method. For example, cDNA encoding thedesired ITF fragment can be used with any method known in the art forproducing recombinant proteins. Exemplary methods are provided herein.ITF fragments, particularly ITF₂₁₋₇₃, can be produced using a Pichiayeast expression system (see, for example, U.S. Pat. Nos. 4,882,279 and5,122,465) transformed with a cDNA encoding long ITF species, such asthe full length ITF (e.g., SEQ ID NO:4) or ITF₁₅₋₇₃, when thefermentation culture is maintained at pH ˜5.0.

[0067] Anti-Inflammatory Agents

[0068] Any suitable anti-inflammatory agent can be formulated with thetrefoil peptide and employed using the method of this invention.Suitable anti-inflammatory agents include, but are not limited tonon-steroidal anti-inflammatory drugs (e.g., ibuprofen, tacrolimus),cyclooxygenase-2-specific inhibitors such as rofecoxib (Vioxx®) andcelecoxib (Celebrex®, topical glucocorticoid agents and specificcytokines directed at T lymphocyte function. Anti-inflammatoryconcentrations known to be effective following rectal administration canbe used. For example, ibuprofen may be present in the composition atconcentrations sufficient to deliver between 25-800 mg per day to thelesion. Corticosteroids may be co-formulated with a trefoil peptide atconcentrations known to be effective for local rectal use.

[0069] Antimicrobial Agents

[0070] Any of the many known antimicrobial agents can be used in thecompositions of the invention at concentrations generally used for theseagents. Antimicrobial agents include antibacterials, antifungals, andantivirals.

[0071] Examples of antibacterial agents (antibiotics) include thepenicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, andamoxicillin), the cephalosporins (e.g., cefadroxil, ceforanid,cefotaxime, and ceftriaxone), the tetracyclines (e.g., doxycycline,minocycline, and tetracycline), the aminoglycosides (e.g., amikacin,gentamycin, kanamycin, neomycin, streptomycin, and tobramycin), themacrolides (e.g., azithromycin, clarithromycin, and erythromycin), thefluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin),and other antibiotics including chloramphenicol, clindamycin,cycloserine, isoniazid, rifampin, and vancomycin.

[0072] Antiviral agents are substances capable of destroying orsuppressing the replication of viruses. Examples of anti-viral agentsinclude 1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide,9−>2-hydroxy-ethoxy methylguanine, adamantanamine,5-iodo-2′-deoxyuridine, trifluorothymidine, interferon, adeninearabinoside, protease inhibitors, thymidine kinase inhibitors, sugar orglycoprotein synthesis inhibitors, structural protein synthesisinhibitors, attachment and adsorption inhibitors, and nucleosideanalogues such as acyclovir, penciclovir, valacyclovir, and ganciclovir.

[0073] Antifungal agents include both fungicidal and fungistatic agentssuch as, for example, amphotericin B, butylparaben, clindamycin,econaxole, fluconazole, flucytosine, griseofulvin, nystatin, andketoconazole.

[0074] Analgesics and Anesthetics

[0075] Any of the commonly used topical analgesics can be used in thecompositions of the invention. The analgesic is present in an amountsuch that there is provided to the distal bowel lesion a concentrationof between one-half and five percent concentration for lidocaine (5-50mg/ml in 20-40 ml per dose of liquid). Examples of other usefulanesthetics include procaine, lidocaine, tetracaine, dibucaine,benzocaine, p-buthylaminobenzoic acid 2-(diethylamino) ethyl ester HCl,mepivacaine, piperocaine, and dyclonine.

[0076] Other analgesics include opioids such as, for example, morphine,codeine, hydrocodone, and oxycodone. Any of these analgesics may also beco-formulated with other compounds having analgesic or anti-inflammatoryproperties, such as acetaminophen, aspirin, and ibuprofen.

[0077] Steroids

[0078] Steroids may be used to treat lesions of the distal bowel. Forexample, ulcerative colitis may be treated using a paste preparation oftriamcinolone (0.1%), hydrocortisone, fluticasone, budesonide, orbeclomethasone.

[0079] 5-Aminosalicylate Derivatives

[0080] 5-aminosalicylate (5-ASA) derivatives are known to be useful fortreating inflammatory bowel diseases such as Crohn's Disease andulcerative colitis. Particularly useful 5-ASA derivatives include, forexample, sulfasalazine, mesalamine, olsalazine, and balsalazide.Sulfasalazine is typically administered as a 3% enema, or orally indoses of 500-1000 mg. Mesalamine is normally administered as a one gramenema, daily for 3-6 weeks, or as a 500 mg suppository, 2-3 times perday for 3-6 weeks. Similar formulations may be prepared for any 5-ASAderivative.

[0081] Production of Intestinal Trefoil Peptides

[0082] Intestinal trefoil peptides and fragments can be produced by anymethod known in the art for expression of recombinant proteins. Nucleicacids that encode trefoil peptides (e.g., human intestinal trefoil(factor (FIG. 4 and 7), human pS2 (FIG. 5 and 8), and human spasmolyticpolypeptide (FIG. 6 and 9) or fragments thereof may be introduced intovarious cell types or cell-free systems for expression thereby allowinglarge-scale production, purification, and patient therapy.

[0083] Eukaryotic and prokaryotic trefoil peptide expression systems maybe generated in which an intestinal trefoil peptide gene sequence isintroduced into a plasmid or other vector, which is then used totransform living cells. Constructs in which the intestinal trefoilpeptide cDNA contains the entire open reading frame inserted in thecorrect orientation into an expression plasmid may be used for proteinexpression. Prokaryotic and eukaryotic expression systems allow for theexpression and recovery of intestinal trefoil peptide fusion proteins inwhich the trefoil peptide is covalently linked to a tag molecule whichfacilitates identification and/or purification. An enzymatic or chemicalcleavage site can be engineered between the trefoil peptide and the tagmolecule so that the tag can be removed following purification.

[0084] Typical expression vectors contain promoters that direct thesynthesis of large amounts of mRNA corresponding to the insertedintestinal trefoil peptide nucleic acid in the plasmid-bearing cells.They may also include a eukaryotic or prokaryotic origin of replicationsequence allowing for their autonomous replication within the hostorganism, sequences that encode genetic traits that allowvector-containing cells to be selected for in the presence of otherwisetoxic drugs, and sequences that increase the efficiency with which thesynthesized mRNA is translated. Stable long-term vectors may bemaintained as freely replicating entities by using regulatory elementsof, for example, viruses (e.g., the OriP sequences from the Epstein BarrVirus genome). Cell lines may also be produced that have integrated thevector into the genomic DNA, and in this manner the gene product isproduced on a continuous basis.

[0085] Expression of foreign sequences in bacteria, such as Escherichiacoli, requires the insertion of an intestinal trefoil peptide nucleicacid sequence into a bacterial expression vector. Such plasmid vectorscontain several elements required for the propagation of the plasmid inbacteria, and for expression of the DNA inserted into the plasmid.Propagation of only plasmid-bearing bacteria is achieved by introducing,into the plasmid, selectable marker-encoding sequences that allowplasmid-bearing bacteria to grow in the presence of otherwise toxicdrugs. The plasmid also contains a transcriptional promoter capable ofproducing large amounts of mRNA from the cloned gene. Such promoters maybe (but are not necessarily) inducible promoters that initiatetranscription upon induction. The plasmid also preferably contains apolylinker to simplify insertion of the gene in the correct orientationwithin the vector.

[0086] Mammalian cells can also be used to express a trefoil peptide.Stable or transient cell line clones can be made using intestinaltrefoil peptide expression vectors to produce the trefoil peptides in asoluble (truncated and tagged) form. Appropriate cell lines include, forexample, COS, HEK293T, CHO, or NIH cell lines.

[0087] Once the appropriate expression vectors are constructed, they areintroduced into an appropriate host cell by transformation techniques,such as, but not limited to, calcium phosphate transfection,DEAE-dextran transfection, electroporation, microinjection, protoplastfusion, or liposome-mediated transfection. The host cells that aretransfected with the vectors of this invention may include (but are notlimited to) E. coli or other bacteria, yeast, fungi, insect cells(using, for example, baculoviral vectors for expression in SF9 insectcells), or cells derived from mice, humans, or other animals. In vitroexpression of trefoil peptides, fusions, or polypeptide fragmentsencoded by cloned DNA may also be used. Those skilled in the art ofmolecular biology will understand that a wide variety of expressionsystems and purification systems may be used to produce recombinanttrefoil peptides and fragments thereof. Some of these systems aredescribed, for example, in Ausubel et al. (Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y. 2000, herebyincorporated by reference).

[0088] Transgenic plants, plant cells and algae are also particularlyuseful for generating recombinant intestinal trefoil peptides for use inthe methods and compositions of the invention. For example, transgenictobacco plants or cultured transgenic tobacco plant cells expressing anintestinal trefoil peptide can be created using techniques known in theart (see, for example, U.S. Pat. Nos. 5,202,422 and 6,140,075).Transgenic algae expression systems can also be used to producerecombinant intestinal trefoil peptides (see, for example, Chen et al.,Curr. Genet. 39:365-370, 2001).

[0089] Once a recombinant protein is expressed, it can be isolated fromcell lysates using protein purification techniques such as affinitychromatography. Once isolated, the recombinant protein can, if desired,be purified further by e.g., high performance liquid chromatography(HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry AndMolecular Biology, Work and Burdon, Eds., Elsevier, 1980).

[0090] Polypeptides of the invention, particularly short intestinaltrefoil peptide fragments can also be produced by chemical synthesisusing, for example, Merrifield solid phase synthesis, solution phasesynthesis, or a combination of both (see, for example, the methodsdescribed in Solid Phase Peptide Synthesis, 2nd ed., 1984, The PierceChemical Co., Rockford, Ill). Optionally, peptide fragments are then becondensed by standard peptide assembly chemistry.

[0091] Dosages

[0092] All of the therapeutic agents employed in the topicalcompositions of the present invention, including the trefoil peptidecomponent, can be used in the dose ranges currently known and used forthese agents. The following are illustrative examples of dose ranges forthe active ingredients of the compositions of the invention. Differentconcentrations of either the trefoil peptide or the other agents may beemployed depending on the clinical condition of the patient, the goal oftherapy (treatment or prophylaxis), and anticipated duration or severityof the damage for which the agent is being given. Additionalconsiderations in dose selection include: disease etiology, patient age(pediatric, adult, geriatric), general health and comorbidity.

[0093] The following examples are intended to illustrate the principleof the present invention and circumstances when trefoil peptide therapyis indicated. The following examples are not intended to be limiting.

EXAMPLE 1 Methods for Treating Proctitis, Enteritis, or MucositisAssociated with Antineoplastic Therapy

[0094] Antineoplastic therapy, including chemotherapy and radiotherapy,can damage the intestinal epithelium, resulting in proctitis, enteritis,or mucositis. Damage to the intestinal mucosa is especially prevalentwhen wide area radiotherapy is delivered to the abdomen for thetreatment of, for example, colorectal, cervical, uterine, ovarian, orprostate cancer. Therapeutic amounts of trefoil peptides can beadministered either previous to, concurrent with, or subsequent toantineoplastic therapy and can be delivered, for example, as an enema orby rectal suppository. Trefoil peptide therapy that followsantineoplastic therapy should begin within the first 14 days after thefinal antineoplastic treatment, preferably within the first 7 days, morepreferably within the 3 days, even more preferably within the first dayand most preferably, immediately following said final antineoplastictreatment.

[0095] Alternatively, intestinal trefoil peptide therapy can beadministered concurrent to the antineoplastic therapy regime. Effectiveconcurrent therapy consists of trefoil peptide administration within 12hours, 6 hours, 3 hours, or simultaneously with every antineoplastictreatment.

[0096] Intestinal trefoil peptide therapy can also begin prior toinitiation of the antineoplastic therapy regime. Pretreatment with atrefoil peptide is prophylactic, thereby mitigating the loss ofintestinal epithelial cells which normally occurs as a consequence ofcancer therapy. Trefoil peptide therapy normally begins 14 days, 7 days,3 days, or 1 day prior to beginning an antineoplastic therapy.

[0097] The most preferred embodiment of the present method consists ofcontinuous trefoil peptide therapy which is begins with a pretreatmentphase, prior to the initiation of antineoplastic therapy, and continuesconcurrently and subsequently to the cancer therapy.

[0098] Rectal administration of a therapeutically effective amount of atrefoil peptide composition using the method of the present invention isdone between once and six times each day, as clinically indicated.Further, clinical indications may warrant supplementation of rectaltrefoil peptide therapy with oral administration of the same or anothertrefoil peptide, or even another intestinal healing agent. Likewise,clinical indications may necessitate the addition of one or moretherapeutic agents, for example, antimicrobials, analgesics, andanti-inflammatories. Additional medicaments can be co-formulated withthe trefoil peptides, or may be administered separately.

EXAMPLE 2 Methods for Treating Mucosal Damage Caused by a PhysicalInjury

[0099] Compositions containing a trefoil peptide, are used to lessencomplications and speed healing of the wound created by the surgicalprocedure or other traumatic injury. These injuries can be caused byprocedures like, for example, a biopsy, a hemorrhoidectomy, or a bowelresection. An aqueous based enema, gel, paste, or suppository, asdescribed above, is applied immediately following the procedure orinjury. Alternatively, a more concentrated medication can be directlyapplied to the wound via a pledget with a stick applicator. Preferably,the trefoil peptide is applied immediately following the surgicalprocedure and then every six hours until epithelial healing is complete.Treatment with a trefoil peptide prior to surgical intervention canspeed post-operative mucosal healing.

EXAMPLE 3 Methods for Treating Crohn's Disease

[0100] Crohn's Disease can be treated using a combination of rectallyadministered ITF and intravenously administered infliximab (Remicade®)).Infliximab is administered by a single intravenous infusion of 5 mg/kg,over about 2 hours. The patient self-administers one suppository,containing one gram of ITF, every second day, beginning 4 days prior toinfliximab treatment. ITF therapy, using the suppositories, is continuedevery second day for six weeks, or as clinically indicated. Patientshaving severe Crohn's disease may require additional infliximab therapytwo weeks and six weeks after the first infusion.

[0101] Other Embodiments

[0102] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

1 9 1 73 PRT Homo sapien 1 Met Leu Gly Leu Val Leu Ala Leu Leu Ser SerSer Ser Ala Glu Glu 1 5 10 15 Tyr Val Gly Leu Ser Ala Asn Gln Cys AlaVal Pro Ala Lys Asp Arg 20 25 30 Val Asp Cys Gly Tyr Pro His Val Thr ProLys Glu Cys Asn Asn Arg 35 40 45 Gly Cys Cys Phe Asp Ser Arg Ile Pro GlyVal Pro Trp Cys Phe Lys 50 55 60 Pro Leu Gln Glu Ala Glu Cys Thr Phe 6570 2 84 PRT Homo sapien 2 Met Ala Thr Met Glu Asn Lys Val Ile Cys AlaLeu Val Leu Val Ser 1 5 10 15 Met Leu Ala Leu Gly Thr Leu Ala Glu AlaGln Thr Glu Thr Cys Thr 20 25 30 Val Ala Pro Arg Glu Arg Gln Asn Cys GlyPhe Pro Gly Val Thr Pro 35 40 45 Ser Gln Cys Ala Asn Lys Gly Cys Cys PheAsp Asp Thr Val Arg Gly 50 55 60 Val Pro Trp Cys Phe Tyr Pro Asn Thr IleAsp Val Pro Pro Glu Glu 65 70 75 80 Glu Cys Glu Phe 3 106 PRT Homosapien 3 Glu Lys Pro Ser Pro Cys Gln Cys Ser Arg Leu Ser Pro His Asn Arg1 5 10 15 Thr Asn Cys Gly Phe Pro Gly Ile Thr Ser Asp Gln Cys Phe AspAsn 20 25 30 Gly Cys Cys Phe Asp Ser Ser Val Thr Gly Val Pro Trp Cys PheHis 35 40 45 Pro Leu Pro Lys Gln Glu Ser Asp Gln Cys Val Met Glu Val SerAsp 50 55 60 Arg Arg Asn Cys Gly Tyr Pro Gly Ile Ser Pro Glu Glu Cys AlaSer 65 70 75 80 Arg Lys Cys Cys Phe Ser Asn Phe Ile Phe Glu Val Pro TrpCys Phe 85 90 95 Phe Pro Asn Ser Val Glu Asp Cys His Tyr 100 105 4 222DNA Homo sapien 4 atgctggggc tggtcctggc cttgctgtcc tccagctctg ctgaggagtacgtgggcctg 60 tctgcaaacc agtgtgccgt gccagccaag gacagggtgg actgcggctacccccatgtc 120 acccccaagg agtgcaacaa ccggggctgc tgctttgact ccaggatccctggagtgcct 180 tggtgtttca agcccctgca ggaagcagaa tgcaccttct ga 222 5 255DNA Homo sapien 5 atggccacca tggagaacaa ggtgatctgc gccctggtcc tggtgtccatgctggccctc 60 ggcaccctgg ccgaggccca gacagagacg tgtacagtgg ccccccgtgaaagacagaat 120 tgtggttttc ctggtgtcac gccctcccag tgtgcaaata agggctgctgtttcgacgac 180 accgttcgtg gggtcccctg gtgcttctat cctaatacca tcgacgtccctccagaagag 240 gagtgtgaat tttag 255 6 390 DNA Homo sapien 6 atgggacggcgagacgccca gctcctggca gcgctcctcg tcctggggct atgtgccctg 60 gcggggagtgagaaaccctc cccctgccag tgctccaggc tgagccccca taacaggacg 120 aactgcggcttccctggaat caccagtgac cagtgttttg acaatggatg ctgtttcgac 180 tccagtgtcactggggtccc ctggtgtttc caccccctcc caaagcaaga gtcggatcag 240 tgcgtcatggaggtctcaga ccgaagaaac tgtggctacc cgggcatcag ccccgaggaa 300 tgcgcctctcggaagtgctg cttctccaac ttcatctttg aagtgccctg gtgcttcttc 360 ccgaagtctgtggaagactg ccattactaa 390 7 3280 DNA Homo sapien 7 atgctggggc tggtcctggccttgctgtcc tccagctctg ctgaggagta cgtgggcctg 60 tgtgagtact gccctgactgccccggtggc agggtgggcg tgaagggaag ggatccagga 120 taagggggga ttctgcattcatttaataat ggccacctgt cacatataca ctttttcctg 180 cgctagccct ttgaagtgggtctttattgt ccccatttca cagacaagga aaccgaggct 240 cagagaaagt taacaacttatccaaggcag ccctgcccag tctgtgttga aatcagggtt 300 tgagcctgag cccatcccctatgaccccat agccatcttt gctggagatt tctaaattac 360 aatataggtc tttatgcattgttccacatt tacaaagaaa aaggaaagat gcaggagaaa 420 aaccctgact tcagaacactgtcaataccg gcaggcacaa ggttcattta gccattgcat 480 agcaaccctg ccatggggtgtggctgctcc attaacccaa gtttgaagga atgagggcat 540 ggcttttatc tgggtgtcttctgagcaggg tcaaaggcag tggttcccga acttgcagcc 600 cattagaatc acctggagagctttaaaaat cctaatgctt ggggcacacc agttacatca 660 gggcatctcc aggcaagatccaggcctcag ctgttttgtt ttgagatagc cttgctttgt 720 cactcactgc tggagtgcagtggcacaatc tcagctcact gcaacctccg cctcctgggt 780 tcaagcaatt cttgtgcctcggcttcaagt agctgggatt acaggcatgc accaccatgc 840 ccagctaatt ttttggatttttagtagaga tggagtttcg ctatgttggc caagctggtc 900 tcaaactcct ggcctcaagtgatcctcctg ccttggcctc ccaaagtgct ggaattacag 960 gtgtaagcca ccatgcccagccaacgtcag tcatttttaa agctctgcag ctgattccag 1020 tgtgagcgaa gtttggatgccaggaggata agcaattacg gactgggagc aagagaaggg 1080 aatgtaagac actgcacgtgattgccattt tcctaaggaa atactcagtt cgttaatgaa 1140 acgcagtgaa cttctgctgcacatacagac atagaggctt gcctgaaaca tgaaaatatt 1200 ggggactgaa ggatgtcccgggagggtggg acatgctcaa caattcagga aggggagatg 1260 cagaaaaaag tgaaaagcaggcagcatgcg ttgcaatgat ctctatggcg tgtgcctctc 1320 ctgtcacggt tttcatttaaaacaaagggg caaggttttg ttggtcaaac aatgaagggt 1380 aactttgttt ctgggttcaagggaccccag attccccagg ggttcctgcc agctggaagg 1440 tacccaggtc cgtatgtgacttcccgagaa ggtgataaga gcgtgccaag gagaaagaca 1500 cttaggcaaa tggccagagtccccgagctg agcatttaac agactgcctc tctttaaata 1560 ttcacaggga aagtgcatcttcctaagggc gagggtttca gcagtggttg aactcggcgg 1620 ggtggggcgg agcgggaggatgcaaacttg caaagtgaag caaacacact caccgcagcc 1680 cagcaagggc tctggcagctgacagggctt tgtctgggac agctgcaaac cagtgtgccg 1740 tgccagccaa ggacagggtggactgcggct acccccatgt cacccccaag gagtgcaaca 1800 accggggctg ctgctttgactccaggatcc ctggagtgcc ttggtgtttc aagcccctgc 1860 aggaagcagg taaggccccagtggcatcgt ggtctgggcc cagccccata aggcaggggg 1920 tctcagggcc tccctgtcctttctgggctg gagatggagg cacaaggacc ccaggaagcc 1980 acacacacac acctgttccaaggcctcaga gcagaggctt cacacttagg gcagccatgg 2040 ccaggggctg tcctcttctgtcccctttat gtaaaacata aaagcaattg tttcaaaaag 2100 gtgttcaaaa tgatggcatcgcatagaggg aactgattta gtaactattc ttgagagaag 2160 tggaaacgca taggtgtggaaagccgggcc gacttttggg ctgtttttgc aaatcggccc 2220 cccagagtct tgtcatttgtggcatcccct acacagacgg caggcggtcc cagccctaga 2280 cgtcaggcct cggtgccacaccccacctcc cccactctgc cccccacaag ggtcatctcc 2340 tctccctctc tctgccgtggtggagggcag gtgcagggca accaccctgg gggttccctc 2400 cccaggggcg gagagcctgcgtgctgtgcg ggtaacagat ggccctgcac acgggtttgc 2460 caccctggct ccaccaggcttagctgcccc acatcgtggg tggggcgatt ggctataagc 2520 catctgccat gtccaagtgccagctcagcc cccacgaagg ccgcacctgc gtgaggtacc 2580 ttcctggaac cagcatccagaggggcctct cttgcccttt gtcctagggt gaaatgcggg 2640 aggctgagtc ctgctggccccggctccctg atcaatgatg ggcccctgcc cagggcctcc 2700 cttcaccctc cccagcaagtccagggtagg ggtgggggtg ggggtccaga gaaggccagg 2760 agagagaggg gtctggctactgtccactgc cggtcctgtt ccttcagctc cactggaact 2820 acactctcct ctgagtgccagccatggccc tgccaaggcc catctcgctt gttatctgcc 2880 tgatccctgg gtcccactatcttgcttagc aacccgaggt gggaatcttg gctattcccc 2940 catgtggtgg ggactcaacactccccggtg actctgggga ggaggcagca ctaggtgctg 3000 gccttggagc ctgccctgaccttgggaagc tgggcagcgt gggtggagag agactgctca 3060 cacaagcctt tgctctgtttgcagaatgca ccttctgagg cacctccagc tgcccccggc 3120 cgggggatgc gaggctcggagcacccttgc ccggctgtga ttgctgccag gcactgttca 3180 tctcagcttt tctgtccctttgctcccggc aagcgcttct gctgaaagtt catatctgga 3240 gcctgatgtc ttaacgaataaaggtcccat gctccacccg 3280 8 4623 DNA Homo sapien 8 9 4252 DNA Homosapien 9 atccctgact cggggtcgcc tttggagcag agaggaggca atggccaccatggagaacaa 60 ggtgatctgc gccctggtcc tggtgtccat gctggccctc ggcaccctggccgaggccca 120 gacaggtaag gcgtgcttct tcctgctctg tggggccaca gccagctctggcagcctccg 180 ccaggagcca ctgttttaca tacatatttt tgagcacctg ttttgtgccaggtgctgttc 240 taggccctta aaagtatatc caatttacag gatcggcaaa agcaggtggagagtaactca 300 gggtggcagg gcccccggag accttcgaga agtgcgacga ggagggggctgccttcagtc 360 ggggctgttt tcctgtgtta ggaagactat acaatcctcc caagtgtcatgtttcaaaga 420 ggaagtgttg gcgtggggtc tcagaatagt gcttttgact gttcatgccaacatctcccc 480 caggggcaga ccctcccaag gcccatccag ataggcccaa atgccggtcccagtgatggc 540 cacctgggag accctctccc acaggcccga atgcccgtcc cagtggtggccaactgggag 600 accctctcct acaggttcct gggctcccct gggatccatg ctctgggagtcaaagccacc 660 tctctcatga gtgcgtggct ggcaacccat attccctggt gttgtcaagtggatcggttg 720 ccctgggtcc ttctagggag tggaggagga ggccattctt gcttccttgggaagtgtttg 780 catctcaact cctttacctg cagaatggat caacggtctg ccctagggctgtcaggaaat 840 gctgtgtggc agcatctgcg acttgcactt tgccagctgt ggggagctgaataacttatt 900 tgccgttatt aggtacagtt tcaaggtggg ggcaggagaa agggctttctacgtttccaa 960 agcaagggtt tccagagagg cctgaagagg gagcgcccag tggtgctgtccgtgccccca 1020 ctgccctcca gccacctctt gatctctgct gtggggtacc gggcctgaggggtgggcttg 1080 ggcagcgtag aagagcagcc agcattgggc tgcagtggga agacccccaagcccatggca 1140 gggagcgggg gagctttgga acccgagaga ggaagtggcc tcggtgtacagaacgaactg 1200 ggtgggtccc cgtgctggcc acccccaggc ccatctgcct gcgcccttgcccccacccca 1260 gcccccagct ctgccccctg tgctgtggga tcacagaggc cgtggcaaactcccctcccc 1320 accccacaca ccctctggct caaggctcag agcgtctttg cgggtcactcaggtccatga 1380 tcctgttaca actgaaatct agaaaattgt gattacagtt tagtgcattcgtgtgtggaa 1440 accatttcca tttatttcca tcatgcgaca aagacaaagc gggtgggcaagacagagtct 1500 gccggaggca gagcaccggg gctggaaatc ttcctccctg aggaggaaacccccccgacc 1560 cccaggatga tgatcctccc tcaccacggg gcctctcttg acccccacagtgtcccgggg 1620 gtgggcgatg atcaccttca cgtcgcgatg gatccagacc ccaggagggcaaggttccca 1680 tggaagctgc tgggcagcgg gagctgaaca cggatccttc ccagcaagccaggaacactt 1740 tctccaaaga catctcgagg cagtccctga tagcaaagca gacaagagaacagcccctct 1800 cggcctcccc tggggcgccc tcacctgagc cagtgtggcc agactgagttcctcccctcc 1860 tatgccccaa ggcagggaca gggaccggag ggtgctctgg gctcctctttcaccccctgc 1920 tgcaggctgt caaccaccag atcctaatag gttgctttct gagacctttgattccgcgga 1980 gctcagagcc tgaagctctg gtgttagaac ctcttgcata agatcctgcggcagccccca 2040 gccagcccca tctgtccacg tgtcttcctc ctctagatcc ctttcctcactgccctgctt 2100 caagctgttt cacagcttgt accctctgtc ggctcctcct agaccaccccacccggtcct 2160 ctcaccttac ctgcaatggg tttccacctc ctgaacacac ctgggtctctggaatggcct 2220 ttgcccatgc ggctccatct tcacctggtg aacctcctcc tgcagggagcccccctgctt 2280 tgttcaacct gcttgtcatt ggcctctccg gggagtgccc tacccccgtggttaccctgg 2340 gcaccctggg acgatggcct tgcgttgtct cgcacatgtt cttgcctttctcctccatca 2400 gatccttaga ctcttttttt tttttttttg agatggagtc ttgctctgtcactcaggctg 2460 gagtgcaatg gtgcgatctt ggctcactac aacctctgcc tcctgggttcaagtgattct 2520 cctgcctcag cctcccaagt agctgggatt acagacgtgt gccacaatgcccgcctaatt 2580 ttttgtattt ttagtagaga tggggcttca ccattttggt caggctggtcttgaactcct 2640 gacctcaagt gattcacctc cttcagcctc ccaaagtgct gggattacaggcatgagcct 2700 gggcccagat atttagactc ttattaatga cttctctggt tttaatttctgggtctctct 2760 cacctggcac agtgcctggc ttttgccatg ctagctccca cttctcatgcacacaaatgg 2820 tgctcagtaa atatttatgt attgagtaaa atttaataat catttgttgaaattaaaaag 2880 tgaataaata agttacctag aaagatgcaa agtccacaaa cctggggcaccttgcatttt 2940 ccctgagcgt aatgtttgca catcaggatg tgaggaccac gtctccctctcatgtcctga 3000 gggttttata tccgcctcac tggacagttg ctgatgtcat tggagaaggaagctggatgg 3060 gtgtgtgcat gataacatca aggaattcag cccacaactt actttgcttcttacctgtgc 3120 actttcagag acgtgtacag tggccccccg tgaaagacag aattgtggttttcctggtgt 3180 cacgccctcc cagtgtgcaa ataagggctg ctgtttcgac gacaccgttcgtggggtccc 3240 ctggtgcttc tatcctaata ccatcgacgt ccctccagaa ggtatggcctttttatacga 3300 tgggttctga agatttagaa ttagttagaa aagtcattta agactacagaggctctgatc 3360 agcatcacca gctatgcctt tacacagagt cacggccgcc agtggtggtgcaatggggta 3420 gcctgagtca ggctgcattc aggtccagga atagaaaggc agggctaagggacttgggaa 3480 gaaacctgat ttccccccgg cttctcttca catctctaac caaaagcctgggaagagcca 3540 ctgttggtaa cgctttctag cttgcctagg atagaggggg aaggcatgacgaaatctgaa 3600 gacatttcat gtattctttt tttttttttt tttttgaaat ggagtctcgctccgttgccc 3660 ctgagctgga gtgcaatggt gcgatcttgg ctcactgcaa tctctgcctcctgagttcaa 3720 cctcagcttc ctagtagctg agattacagg tgtgtgccac tacgcccagctaaatttttt 3780 ttgtattttt agtatagacg gggtttcacc atgttggcca gaccggtcttgaactcttga 3840 cctcaggtga tctgcccgcc tcagcctccc agagagctgg gattacaggcgtgagccacc 3900 gtgcccggct gacagttcat gttttctaaa gaatgtgcct atggatactttaaagtaaaa 3960 actctgtaat tgtttaaatg tgaaagaaaa tgtttatcct cactaaagcatctctttctc 4020 cctccccctc acccctgtag aggagtgtga attttagaca cttctgcagggatctgcctg 4080 catcctgacg cggtgccgtc cccagcacgg tgattagtcc cagagctcggctgccacctc 4140 caccggacac ctcagacacg cttctgcagc tgtgcctcgg ctcacaacacagattgactg 4200 ctctgacttt gactactcaa aattggccta aaaattaaaa gagatcgatatt 4252

What is claimed is:
 1. A method for treating or preventing a lesion ofthe distal bowel of a mammal comprising administering to the rectum ofsaid mammal a therapeutically effective amount of an intestinal trefoilpeptide.
 2. The method of claim 1, wherein said intestinal trefoilpeptide is spasmolytic polypeptide, pS2, or intestinal trefoil factor.3. The method of claim 2, wherein said intestinal trefoil peptide isintestinal trefoil factor.
 4. The method of claim 1, wherein saidintestinal trefoil peptide is ITF₁₅₋₇₃, ITF₂₁₋₇₃, ITF₁₋₇₂, ITF₁₅₋₇₂, orITF₂₁₋₇₂.
 5. The method of claim 1, wherein said mammal is a human. 6.The method of claim 1, wherein said lesion is enteritis or proctitis. 7.The method of claim 1, wherein said lesion is caused by Crohn's Disease.8. The method of claim 1, wherein said lesion is caused by ulcerativecolitis.
 9. The method of claim 1, wherein said lesion is caused byantineoplastic therapy.
 10. The method of claim 9, wherein saidantineoplastic therapy is radiation therapy.
 11. The method of claim 9,wherein said antineoplastic therapy is chemotherapy.
 12. The method ofclaim 1, wherein said lesion is the result of a hemorrhoidectomy. 13.The method of claim 1, wherein said lesion is the result of a biopsyprocedure or surgical intervention.
 14. The method of claim 13, whereinsaid lesion is the result of tumor resection.
 15. The method of claim 1,wherein said lesion is caused by a bacterial, viral, or fungalinfection.
 16. The method of claim 1, further comprising administeringto said mammal a second therapeutic.
 17. The method of claim 16, whereinsaid second therapeutic agent is an anti-inflammatory agent.
 18. Themethod of claim 16, wherein said second therapeutic agent is rofecoxibor celecoxib.
 19. The method of claim 16, wherein said secondtherapeutic agent is an antibacterial agent.
 20. The method of claim 19,wherein said antibacterial agent is a penicillin, a cephalosporin, atetracycline, or an aminoglycoside.
 21. The method of claim 16, whereinsaid second therapeutic agent is an anti-fungal agent.
 22. The method ofclaim 21, wherein said anti-fungal agent is nystatin or Amphotericin B.23. The method of claim 16, wherein said second therapeutic agent is ananti-viral agent.
 24. The method of claim 23, wherein said anti-viralagent is acyclovir.
 25. The method of claim 16, wherein said secondtherapeutic agent is an analgesic.
 26. The method of claim 25, whereinsaid analgesic is lidocaine or benzocaine.
 27. The method of claim 16,wherein said second therapeutic agent is a steroid.
 28. The method ofclaim 27, wherein said steroid is triamcinolone, budesonide, orhydrocortisone.
 29. The method of claim 16, wherein said secondtherapeutic is sulfasalazine, mesalamine, olsalazine, balsalazide, ormetronidazole.
 30. The method of claim 16, wherein said secondtherapeutic is infliximab.
 31. The method of claim 16, wherein saidtrefoil peptide and said second therapeutic are administered in the sameformulation.
 32. The method of claim 16, wherein said trefoil peptideand said second therapeutic are administered in different formulations.33. The method of claim 32, wherein said trefoil peptide and said secondtherapeutic are administered within 14 days of each other.
 34. Themethod of claim 33, wherein said trefoil peptide and said secondtherapeutic are administered within 24 hours of each other.
 35. Themethod of claim 9, wherein said intestinal trefoil peptide isadministered within 14 days of said antineoplastic therapy.
 36. Themethod of claim 35, wherein said intestinal trefoil peptide isadministered within 24 hours of said antineoplastic therapy.
 37. Apharmaceutical composition suitable for therapeutic delivery to therectum of a mammal, said composition comprising an intestinal trefoilpeptide and a pharmaceutically acceptable carrier.
 38. The compositionof claim 37, wherein said intestinal trefoil peptide is spasmolyticpolypeptide, pS2, or intestinal trefoil factor.
 39. The composition ofclaim 38, wherein said intestinal trefoil peptide is intestinal trefoilfactor.
 40. The composition of claim 37, wherein said intestinal trefoilpeptide is ITF₁₅₋₇₃, ITF₂₁₋₇₃, ITF₁₋₇₂, ITF₁₅₋₇₂, or ITF₂₁₋₇₂.
 41. Thecomposition of claim 37, wherein said composition is a suppository. 42.The composition of claim 37, wherein said composition is an enema. 43.The composition of claim 37, wherein said composition comprisesmicrospheres.
 44. The composition of claim 37, wherein said compositionfurther comprises a mucoadhesive agent.
 45. The composition of claim 37,wherein said composition further comprises a second therapeutic agent.46. The composition of claim 45, wherein said second therapeutic agentis an anti-inflammatory agent.
 47. The composition of claim 45, whereinsaid second therapeutic agent is an antibacterial agent.
 48. Thecomposition of claim 47, wherein said antibacterial agent is apenicillin, a cephalosporin, a tetracycline, or an aminoglycoside. 49.The composition of claim 45, wherein said second therapeutic agent is ananti-fungal agent.
 50. The composition of claim 49, wherein saidanti-fungal agent is nystatin or Amphotericin B.
 51. The composition ofclaim 45, wherein said second therapeutic agent is an anti-viral agent.52. The composition of claim 51, wherein said anti-viral agent isacyclovir.
 53. The composition of claim 45, wherein said secondtherapeutic agent is an analgesic.
 54. The composition of claim 53,wherein said analgesic is lidocaine or benzocaine.
 55. The compositionof claim 45, wherein said second therapeutic agent is a steroid.
 56. Thecomposition of claim 55, wherein said steroid is triamcinolone,budesonide, or hydrocortisone.
 57. The composition of claim 45, whereinsaid second therapeutic is sulfasalazine, mesalamine, olsalazine, orbalsalazide.
 58. The composition of claim 45, wherein said secondtherapeutic is metronidazole.